Teriflunomide shifts the astrocytic bioenergetic profile from oxidative metabolism to glycolysis and attenuates TNFα-induced inflammatory responses

Astrocytes utilize both glycolytic and mitochondrial pathways to power cellular processes that are vital to maintaining normal CNS functions. These cells also mount inflammatory and acute phase reactive programs in response to diverse stimuli. While the metabolic functions of astrocytes under homeostatic conditions are well-studied, the role of cellular bioenergetics in astrocyte reactivity is poorly understood. Teriflunomide exerts immunomodulatory effects in diseases such as multiple sclerosis by metabolically reprogramming lymphocytes and myeloid cells. We hypothesized that teriflunomide would constrain astrocytic inflammatory responses. Purified murine astrocytes were grown under serum-free conditions to prevent acquisition of a spontaneous reactive state. Stimulation with TNFα activated NFκB and increased secretion of Lcn2. TNFα stimulation increased basal respiration, maximal respiration, and ATP production in astrocytes, as assessed by oxygen consumption rate. TNFα also increased glycolytic reserve and glycolytic capacity of astrocytes but did not change the basal glycolytic rate, as assessed by measuring the extracellular acidification rate. TNFα specifically increased mitochondrial ATP production and secretion of Lcn2 required ATP generated by oxidative phosphorylation. Inhibition of dihydroorotate dehydrogenase via teriflunomide transiently increased both oxidative phosphorylation and glycolysis in quiescent astrocytes, but only the increased glycolytic ATP production was sustained over time, resulting in a bias away from mitochondrial ATP production even at doses down to 1 μM. Preconditioning with teriflunomide prevented the TNFα-induced skew toward oxidative phosphorylation, reduced mitochondrial ATP production, and reduced astrocytic inflammatory responses, suggesting that this drug may limit neuroinflammation by acting as a metabolomodulator.


Results
TNFα drives inflammatory signaling and chemokine release in purified primary astrocytes. In order to obtain quiescent astrocytes without microglial contamination, mixed glial cultures were treated with clodrosome 19 and then grown under serum-free conditions for at least 14 days. Culture purity was assessed by flow cytometry (98.8% ± 0.2% ASCA-2 + astrocytes; 0.3% ± 0.1% O4 + oligodendrocytes; 0.06% ± 0.04% CD11b + microglia; 0.9% ± 0.3% unlabeled other). Removing serum from the culture system was critical to preventing a spontaneous reactive state 20 and for establishing reproducible baseline bioenergetic profiles. Under resting conditions, the astrocytes exhibited an elongated morphology (Fig. 1A) marked by a low-complexity GFAP + ramified phenotype (Fig. 1B). Stimulation of these quiescent astrocytes with TNFα (10 ng/mL) for 1 h induced NFκB nuclear translocation (Fig. 1C) and phosphorylation (Fig. 1D), indicating inflammatory pathway activation. TNFα-induced NFκB phosphorylation was four times higher relative to untreated conditions (P = 0.0046). TNFα stimulation for 24 h also induced secretion of Lcn2 (116-fold), CCL5 (226-fold), CCL2 (642-fold), and CXCL2 (36-fold) (Fig. 1E). We conclude that TNFα induces an inflammatory phenotype in pure cultures of astrocytes grown under serum-free conditions. TNFα increases oxidative phosphorylation and biases astrocytes toward mitochondrial ATP production. Previous work has shown that inflammatory cytokine stimulation converges on NFκB activation to drive downstream rewiring of the astrocytic bioenergetic phenotype 21 . To characterize the bioenergetic impact of TNFα on purified astrocytes under serum-free conditions we measured oxidative phosphorylation and ATP production using real-time extracellular flux analysis 22 . Cells stimulated with TNFα (10 ng/mL) for 24 h exhibited increased basal respiration, increased maximal respiration, and increased ATP production relative to vehicle (DMSO)-treated astrocytes using the oxygen consumption rate (OCR) normalized to total cellular protein (pmol/min/μg) ( Fig. 2A, B). Measurement of the normalized extracellular acidification rate (ECAR) (mpH/ min/μg) under the same conditions further revealed that TNFα stimulation increased the glycolytic reserve and glycolytic capacity of astrocytes but did not change the basal glycolytic rate (Fig. 2C, D). Analysis of the normalized ATP production rate (pmol/min/μg) mediated by glycolysis (glycoATP) versus oxidative phosphorylation (mitoATP) showed that TNFα increased both glycolytic and mitochondrial ATP production (Fig. 2E). However, taking the ratio of mitoATP to glycoATP, defined herein as the ATP rate index, shows that TNFα stimulation for 24 h increased overall astrocytic ATP production (Fig. 2F) by preferentially biasing the cell to produce ATP through enhanced oxidative phosphorylation.
TNFα-induced Lcn2 secretion is ATP-dependent. Based on the TNFα-induced bias toward oxidative phosphorylation-derived ATP production, we hypothesized that astrocytic secretion of factors such as Lcn2 in response to TNFα is dependent upon ATP production. Given that glucose metabolism-derived metabolites enter the TCA cycle as pyruvate while glutamine is incorporated into the TCA cycle by conversion to α-ketoglutarate, we tested the TNFα-induced response in a TCA substrate-deficient environment. Astrocytes were incubated in DMEM lacking glucose (basal = 10 mM), pyruvate (basal = 1.3 mM), and glutamine (basal = 4 mM) media for 30 min and then resupplemented with pyruvate (Pyr or P: 0.5 or 1.0 mM), glutamate ( www.nature.com/scientificreports/ 0.1 mM), and/or glucose (Glc or G: 0.1 or 1.0 mM) for 24 h in the presence or absence of TNFα (10 ng/mL) (PEG = Pyr + Glu + Glc). The total amount of cellular ATP in the absence of TCA substrates was decreased from 26,087 units to 37 units and stimulation with TNFα did not alter this difference (Fig. 3A). Notably, the absence of TCA substrates reduced basal secretion of Lcn2 from 58 to 10 pg/mL/10 5 cells and profoundly suppressed TNFα-induced Lcn2 release from 3642 pg/mL/10 5 cells in the resupplemented media to 30 pg/mL/10 5 cells in the deficient media (Fig. 3B). Adding back individual substrates revealed that each factor was sufficient to at least partially restore ATP levels, although glutamate was less effective than either pyruvate or glucose at the concentrations utilized (Fig. 3C). In parallel, each factor was sufficient to restore Lcn2 secretion in response to TNFα stimulation, with pyruvate providing the most robust response (Fig. 3D). The relationship between substrate-induced ATP restoration and TNFα-induced Lcn2 release indicates that the inflammatory response scales with the amount of cellular ATP (R 2 = 0.76) (Fig. 3E). Finally, pharmacological inhibition of oxidative phosphorylation-derived ATP synthesis with oligomycin (1 μM) reduced Lcn2 production in response to TNFα from 3000 pg/mL/10 5 cells to 1281 pg/mL/10 5 cells (Fig. 3F). These findings indicate that TNFα-induced Lcn2 secretion by astrocytes requires ATP and that oxidative phosphorylation is a primary source of this energy substrate.
Teriflunomide biases astrocytic metabolism toward glycolytic ATP production. Based (Fig. 4A). In contrast, glycolytic ATP production was increased at 24 and 72 h in response to 30 μM TF (Fig. 4B). This effect was confirmed by ECAR measurement, which showed that TF increased basal glycolysis and glycolytic capacity in the astrocytes (Supplemental Fig. 1). Moreover, total cellular ATP production rate was increased at both 24 and 72 h in response to either 10 or 30 μM TF (Fig. 4C).
Calculating the ATP rate index at 72 h, as above, revealed that even 1 μM TF trended toward shifting astrocytic metabolism toward glycolytic ATP production relative to DMSO ( Fig. 4D; DMSO vs 1 μM TF, P = 0.0899). This effect was not associated with increased cellular toxicity, as all 3 TF concentrations preserved viability at or above vehicle control levels ( Fig. 4E; 30 μM shown). We conclude that teriflunomide increases both mitochondrial and glycolytic ATP production, but that the predominant effect at 72 h is a skew toward glycolytic production. Indeed, 10 μM TF reduced the ATP rate index to less than 1.3 (relative to almost 4 for the vehicle control),    www.nature.com/scientificreports/ indicating near equalization of the contribution of glycolytic ATP production and oxidative phosphorylation production (Fig. 4D).
Teriflunomide preconditioning blocks the bioenergetic impact of TNFα and reduces the astrocytic inflammatory response. The bias toward glycolytic ATP production induced in astrocytes at 72 h by TF suggested that this inhibitor might counter the oxidative phosphorylation skew driven by TNFα. Using a preconditioning paradigm in which astrocytes were treated with TF (30 μM) at -48 h and then treated again with TF (30 μM) and TNFα (10 ng/mL) at 0 h, revealed that 24 h later the cellular bioenergetic profile was renormalized relative to TNFα alone. While TF alone persistently increased glycoATP and TNFα alone biased toward mitoATP, astrocytes that were preconditioned with TF and then stimulated with TNFα exhibited glycoATP and mitoATP production rates that were not different from vehicle control (Fig. 5A) (glycoATP: T + T vs DMSO, P = 0.1338; mitoATP: T + T vs DMSO, P = 0.5020). This renormalization effect is further evident in the ATP rate index, which shows that TF pushed TNFα-induced ATP production back to control levels ( Fig. 5B) (ATP rate index: T + T vs DMSO, P = 0.8638). TF preconditioning also blocked the TNFα-induced increase in oxidative phosphorylation (Fig. 5C), with normalization of basal respiration, maximal respiration, and ATP production to vehicle control levels (basal respiration: T + T vs DMSO, P = 0.8059; maximal respiration: T + T vs DMSO, P = 0.9588; ATP: T + T vs DMSO, P = 0.9935). The TF effect was not mediated by inhibition of NFκB, as phosphorylation and nuclear translocation of this signaling molecule were unchanged relative to TNFα alone (Fig. 5D, E). Notably, phosphorylation of p38 MAPK in response to TNFα was completely prevented by TF preconditioning (Fig. 5D). Finally, TF preconditioning significantly reduced the inflammatory response to TNFα, with a 40% reduction in Lcn2 secretion, a 40% reduction in CCL5, a 25% reduction in CCL2, and a 15% reduction in CXCL2 (Fig. 5F). These results indicate that teriflunomide renormalizes the astrocytic bioenergetic response to TNFα, resulting in a reduced inflammatory response.  . Mitochondrial ATP production rate (A), glycolytic ATP production rate (B), and total ATP production rate (C) were measured using the Seahorse XF analyzer. Data are normalized to total protein (μg) per well. Each symbol represents one well from 3 separate experiments, shown in nested layout. Mean ± SEM is shown for each biological replicate. P-values were calculated using mixed model nested ANOVA between treatment conditions. (D) The ratio of mitochondrial ATP production rate to glycolytic ATP production rate was calculated from the data in (A) and (B) to indicate the ATP rate index in the 72 h paradigm. Each symbol represents one well from 3 separate experiments, shown in nested layout. Mean ± SEM is shown for each biological replicate. P-values were calculated using mixed model nested ANOVA between treatment conditions. (E) Viability of astrocytes exposed to teriflunomide (

Discussion
Immunomodulation via metabolic reprogramming and control of bioenergetic pathways has revolutionized our approach to therapy in numerous immune-mediated diseases 23 . Notably, however, many of these therapies impose control by inhibiting resources required for the robust proliferative response induced by inflammatory and autoimmune drivers 23,24 . For example, teriflunomide-induced inhibition of DHODH limits de novo pyrimidine biosynthesis and thereby reduces T cell proliferation 25 , resulting in improved outcomes in patients with relapsing-remitting multiple sclerosis 18 . Teriflunomide also depletes ATP in highly proliferative cells 26 and interferes with complex III of the mitochondrial electron transport chain, reducing T cell activation in response to proliferative drivers 18 . Indeed, most immune cells, per se, share a common proliferative response to inflammatory activators that results in a shift from basal oxidative metabolism to aerobic glycolysis [27][28][29] .
In contrast to immune cells such as lymphocytes, which have relatively minimal basal metabolic demands prior to induction of proliferation and activation 30 , astrocytes under homeostatic conditions bear a large and critical metabolic burden 31 . Astrocytes support the energy demands of neurons and neural circuits by providing lactate generated through the metabolism of glucose to pyruvate in the glycolytic pathway. Astrocytes also support neurotransmitter homeostasis and network function via the ATP-dependent conversion of extracellular glutamate into glutamine that is released for neuronal uptake and reprocessing 32 . At the same time, astrocytes respond to inflammatory drivers using mechanisms that are shared with immune cells 33 . However, it is only under the most profound injury conditions that astrocytes proliferate in response to these drivers 34 . This dichotomy suggests that metabolic modulation of astrocytes may involve processes and responses that are fundamentally different from immune cells such as lymphocytes.
Exploring the specific immunometabolism of astrocytes requires careful consideration of the system used for analysis. To date, the majority of work on astrocyte metabolism has involved two problematic components that limit interpretability and mechanistic understanding. The first issue is the use of FBS as a source of trophic support in glial cultures. Given that FBS itself drives inflammatory activation of astrocytes 35 , the analysis of metabolic pathways induced by inflammatory drivers in the presence of FBS is compromised by the absence of a metabolically quiescent baseline. The second problem is the assignment of inflammatory and metabolic responses to astrocytes in culture models that are better characterized as mixed glia 36 . Most studies employ shake-off cultures that enrich astrocytes by physically displacing microglia and oligodendrocytes from a firmly adherent astrocyte monolayer. However, this method only results in a reduction in microglia, not complete ablation. Therefore, in this study we have effectively completely ablated microglia by treating post-shake-off cultures with clodrosome 19 and we have cultured the highly purified astrocytes (> 98.8% ASCA-2 + astrocytes, < 0.1% CD11b + microglia, < 0.3% O4 + oligodendrocytes) in long-term serum-free conditions that promote a quiescent baseline.
Using this system, we demonstrated that stimulation with TNFα drives astrocytic production of Lcn2 and other inflammatory factors and simultaneously increases both oxidative phosphorylation and glycolysis, with a robust skew toward mitochondrial ATP production. We further showed that the Lcn2 response induced by TNFα is dependent on ATP. However, blocking the electron transport chain with oligomycin only attenuated Lcn2 release by approximately 50%, indicating that astrocytes maintain a glycolytic component of ATP production even under inflammatory drive. The observation that ATP production and the Lcn2 response to TNFα is reconstituted, at least in part, by reintroduction of either pyruvate, glutamate, or glucose indicates that astrocytes have a high degree of metabolic substrate flexibility. Despite the homeostatic energy differences between astrocytes and lymphocytes and the differential proliferative response induced by inflammation, these findings suggest that both cell types engage oxidative phosphorylation-dependent ATP production as part of the response to inflammatory drivers.
Under quiescent conditions, we found that astrocytes generate approximately 20 pmol/min/μg ATP from oxidative phosphorylation and approximately 10 pmol/min/μg via glycolysis. Incubation of these homeostatic cells with teriflunomide for 24 h increased ATP production via both pathways in a dose-dependent manner, doubling the mitochondrial ATP output to about 40 pmol/min/μg and more than doubling the glycolytic output to about 25 pmol/min/μg. In itself, this suggests that teriflunomide induces an overall enhancement of astrocytic bioenergetic output, a finding that may have relevance to therapeutic interventions in neurodegenerative conditions associated with compromised ATP production 37,38 . However, the impact of teriflunomide on oxidative phosphorylation was not maintained when cells were stimulated with the drug and then restimulated 48 h later and assessed at 72 h after the initial exposure. Instead, this paradigm resulted in continued enhancement of glycolytic ATP production, increasing the rate to 30 pmol/min/μg. Calculating the ratio between mitochondrial and glycolytic ATP production rate revealed that teriflunomide skewed the basal metabolism of astrocytes from a state in which oxidative phosphorylation output dominated glycolytic output by over 2:1 to a state in which glycolytic output equaled mitochondrial production. Moreover, this effect occurred even at low doses and was associated with increased glycolytic capacity and glycolytic reserve but unchanged basal or maximal respiration. This outcome is sharply different from the naive T cell response to teriflunomide, in which the drug does not alter mitochondrial respiration or glycolytic capacity over 72 h 18 .
Teriflunomide also robustly altered the astrocytic bioenergetic response to TNFα stimulation, though the nature of this effect was different from the lymphocyte response to the drug. Teriflunomide essentially renormalized ATP production in astrocytes, returning both glycolytic and mitochondrial rates to below baseline levels and restoring the ratio of mitochondrial to glycolytic production to more than 2:1. The drug also returned basal and maximal respiration to baseline levels. In contrast, teriflunomide reduced these measures in stimulated T cells but did not return the cells to baseline levels 18 . Unexpectedly, despite the nearly complete renormalization of metabolism, teriflunomide only partially inhibited Lcn2 and chemokine production induced by TNFα stimulation. Notably, despite evidence that TF inhibits NFκB signaling in some contexts 16   www.nature.com/scientificreports/ TF completely inhibited p38 phosphorylation in response to TNFα stimulation and reduced phospho-p38 levels below baseline in unstimulated astrocytes. Given the TF-induced skew in basal metabolism toward enhanced glycolytic ATP production under non-inflammatory conditions and the induction of mitochondrial ATP production in response to TNFα, these findings suggest that p38 activation may depend upon oxidative phosphorylation. The p38 serine-threonine kinase is a mitogen-activated protein kinase (MAPK) that is proximally activated by the dual-specificity MAPK kinases MKK3 and MKK6 39 . p38 is a critical regulator of cellular stress responses, driving specific signaling cascades involved in activation, differentiation, apoptosis, and autophagy in cells stressed by cytokines, radiation, osmotic shock, heat shock, and oxidative stress 40 . These cascades converge on MKK3/6 and p38 downstream from a diverse range of MAPK kinase kinases. One such upstream kinase is the apoptosis signal-related kinase ASK1, which is activated by both oxidative stress 41 and TNFα 42 via increased intracellular reactive oxygen species (ROS) production 43 . TNFα-induced mitochondrial ATP production results in generation of ROS 44 , oxidation of thioredoxin 45 , and consequent derepression of ASK1 activity 46 , leading to p38 activation 42 . In turn, the primary downstream target of p38, the MAPK-activated protein kinase 2 (MAP-KAPK2), is involved in post-transcriptional regulation of numerous inflammatory and stress response mRNAs. The likely mechanism for post-transcriptional control by MAPKAPK2 is stabilization of mRNAs bearing adenosine/uridine-rich repeat elements (AREs) such as AUUUA 47 -in the absence of MAPKAPK2 signaling these AREs destabilize transcripts and shorten half-life, reducing the pool of mRNA available for translation 48 . Notably, Lcn2 has a canonical AUUUA motif within an intron that is conserved across splice variants 49, 50 . In this context it is therefore interesting that TF-treated astrocytes failed to show a sustained increase in Lcn2 RNA after stimulation for 24 h with TNFα (TNFα only = sixfold induction; TNFα + TF = 0.9-fold induction). Given the incomplete inhibition of Lcn2 release in TF-treated astrocytes, this suggests that an initial wave of inflammatory factor release occurred in response to TNFα, but continued production and release was thwarted by degradation of the RNA encoding these factors. Furthermore, the inability to maintain the inflammatory response due to RNA degradation is consistent with the observation that TF does not prevent NFκB activation. Thus, in TF-treated astrocytes stimulation with TNFα may drive signaling through NFκB, engaging the machinery necessary to generate inflammatory mRNAs, but the absence of p38 signaling results in failure to stabilize these RNAs, thereby limiting translation and production of additional protein. Further research is necessary to test this hypothesis, but one intriguing speculation that arises from this concept is that TF may serve to more broadly impede inflammatory responses associated with p38 signaling, such as the senescence-associated secretory pathway or neuroinflammation associated with seizures and psychiatric disorders.
In conclusion, using highly purified astrocytes grown under conditions that preserve a non-reactive state, we found that TNFα drives inflammatory signaling and biases the cellular bioenergetic profile toward mitochondrial ATP production. In contrast, teriflunomide biases astrocytic metabolism toward glycolytic ATP production. Preconditioning with teriflunomide prevents the TNFα-induced bioenergetic shift, resulting in maintenance of homeostatic ATP production. Consequently, the astrocytic inflammatory response is reduced. These findings suggest that teriflunomide impacts metabolism in astrocytes in a manner that may reduce neuroinflammation across a spectrum of neurological diseases.

Mice.
No experiments were performed on live animals. C57Bl/6 mice were used for preparation of primary astrocytes, as described below. Neonatal animals were not sexed prior to euthanasia. Neonatal mice were killed by decapitation, as per the AVMA Guidelines for the Euthanasia of Animals and the ACLAM Task Force on Rodent Euthanasia. All studies were conducted in accordance with the United States Public Health Service's Policy on Humane Care and Use of Laboratory Animals and were approved by the Mayo Clinic Institutional Animal Care and Use Committee (Animal Welfare Assurance number A3291-01). Where relevant, experiments and procedures conformed to ARRIVE guidelines. Figure 5. Teriflunomide preconditioning blocks the bioenergetic effect of TNFα and inhibits release of reactive inflammatory factors. Quiescent astrocytes were preconditioned with teriflunomide (TF; 30 μM) using the 72 h treatment paradigm. TNFα (10 ng/mL) was added at the same time as the final teriflunomide treatment and glycolytic and mitochondrial ATP production (A), the ATP rate index (B), and the oxygen consumption rate (C) were measured 24 h later. T + T = TF + TNFα condition. Each symbol represents one well from 3 separate experiments, shown in nested layout. Mean ± SEM is shown for each biological replicate. P-values were calculated using mixed model nested ANOVA between treatment conditions. TF reduced the TNFαinduced increase in all assays to control (DMSO) levels (glycoATP: T + T vs DMSO, P = 0.1338; mitoATP: T + T vs DMSO, P = 0.5020; basal respiration: T + T vs DMSO, P = 0.8059; maximal respiration: T + T vs DMSO, P = 0.9588; ATP: T + T vs DMSO, P = 0.9935). Preconditioning with teriflunomide did not inhibit NFκB phosphorylation (D) or nuclear translocation (E) in response to TNFα stimulation. Immunoblots for NFκB derive from the same transfer membrane. Immunoblots for p38 derive from the same membrane. Uncropped blots are available in the Supplementary Information. p38 phosphorylation was completely abrogated by teriflunomide preconditioning. Upper panels in (E) show GFAP (green), NFκB (red), and DAPI (blue); lower panels show specific NFκB signal. (F) Teriflunomide preconditioning reduced Lcn2, CCL5, CCL2, and CXCL1 release from astrocytes stimulated with TNFα (10 ng/mL). Each symbol represents one well from 3 separate experiments, shown in nested layout. Mean ± SEM is shown for each biological replicate. P-values were calculated using mixed model nested ANOVA between treatment conditions. Scale bars are 20 μm. www.nature.com/scientificreports/ Mouse cortical primary astrocyte cultures. Primary murine astrocyte cultures were prepared from P1-P3 C57BL/6J mouse pups, as described 51 . Briefly, pups were individually separated from the dam, wiped quickly with 70% EtOH, and decapitated. The skull was opened with forceps and the brain was removed into PBS. The olfactory bulbs and cerebellum were removed, the cortices were separated, the inner face of each cortex was scooped clean with forceps, and meninges were removed from the cortical surface. After collecting all cortices, the pooled material was washed in PBS, minced into 1 mm 2 blocks with a sterile single-edge blade, and digested with 0.25% trypsin (Invitrogen, 15090-046, no phenol red) prepared in Earle's balanced salt solution (EBSS; NaCl 7.02 g/L, NaH 2 PO 4 -H 2 O 0.84 g/L, KCl 0.14 g/L, glucose 3.60 g/L, NaHCO 3 0.84 g/L) containing HEPES (4.80 g/L) and bovine serum albumin (BSA; 3.00 g/L) for 30 min at 37 °C with shaking at 80 rpm. Trypsin was quenched with 10% heat-inactivated fetal bovine serum (FBS) and the suspension was incubated on ice for 5 min with 0.08% MgSO4 and DNase I (0.02 mg/mL). Tissue pieces were pelleted at 200 g for 5 min, triturated into a single cell suspension, resuspended in EBSS containing HEPES and BSA, underlaid with 1.25 mL 4% BSA prepared in EBSS, and centrifuged at 100 g for 8 min without braking. The cell pellet was resuspended in astrocyte growth media (AGM; high glucose Dulbecco's modified eagle medium (DMEM, VWR, 45000-304); 10% FBS (Sigma, 12306C); 1% penicillin-streptomycin (Gibco, 15,140-122)) and cells were plated at 1.3 × 10 5 cells/cm 2 on T75 flasks coated with poly-l-lysine hydrobromide (Sigma, P1524  (3-(4,5-Dimethlythiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) viability assay. Cells (8 × 10 4 ) were plated in a 48-well dish and allowed to reach confluency. Light-sensitive yellow tetrazolium MTT (3-(4,5-dimethlythiazolyl-2)-2,5-diphenyltetrazolium bromide, Sigma, M2128) was then added at a 1:10 dilution cells were incubated at 37 °C, 5% CO 2 for 1 h. Metabolically active cells reduced the MTT through interaction with NADH and NADPH, resulting in formation of an intracellular purple formazan precipitate. After aspiration of the media using a fine tip, 400 µL of SDS-DMF was added to lyse the cells and solubilize the formazan. After pipetting up a down, 100 µL of each sample was transferred to each of 2 duplicate wells of a 96-well plate and placed on a shaker for 15 min to promote dissolution. Formazan was quantified by spectrophotometric readings at 570 nm on a SpectraMax M3 spectrophotometer (Molecular Devices) using SoftMax Pro Software.
Adenosine triphosphate (ATP) detection assay. ATP  www.nature.com/scientificreports/ were pre-equilibrated one day before extracellular flux analysis using XF calibrant (pH 7.4; Agilent #100840-000) at 37 °C in a non-CO 2 incubator. On the day of the experiment, we added XF-DMEM media (Agilent #103575-100) containing 180 µM sodium pyruvate (Sigma #S8636), 0.5 mM glutamine (Gln; Gibco #2503008), 5 µM glutamate (Glu; Sigma #PHR2634), and 10 mM glucose (Sigma #G8769; for mito stress only, as the glycolysis stress test delivers the same concentration of glucose during the assay) to the cells just before incubation at 37 °C for 1 h. For glycolysis stress analysis, glucose (10 mM), oligomycin (3 µM; Sigma #75351) and 2-DG (50 mM; Cayman #14325) were injected via port A, B and C respectively. For the mitochondrial stress assay oligomycin (3 µM), FCCP (1.5 µM; Sigma #C2920) and rotenone (Sigma #R8875) + antimycin A (Sigma #A8674) (1 µM + 1 µM) were injected via port A, B and C respectively. Real-time ATP production rate was assessed by analyzing extracellular flux in response to 3 µM oligomycin and 1 µM rotenone + antimycin A (1:1) injected via port A and B respectively. Total protein per well was quantified by BCA analysis after the seahorse experiment and these values were used to normalize the bioenergetic measurements. All analyses were performed in Wave software and then exported to Excel and GraphPad Prism 8.0 for further analysis.
Experimental design and statistical analysis. Experiments were performed in at least 3 separate biological replicates (cultures) prepared from independent animal cohorts. Where applicable, a minimum of 3 technical replicates were utilized in each experiment. A nested biological:technical replicate design was employed for all relevant graphs and statistical analyses. Mixed model t-tests were used to calculate significance for experiments with one treatment; mixed model one-way ANOVA was used to for nested designs with multiple treatments. Data from each biological replicate are presented as mean ± SEM of the technical replicates. P-values less than 0.05 were considered significant. Both Microsoft Excel and GraphPad Prism 8 (GraphPad Software, Inc.) were used to analyze the data.
Ethics approval. All studies were conducted in accordance with the United States Public Health Service's Policy on Humane Care and Use of Laboratory Animals and with approval from the Mayo Clinic Institutional Animal Care and Use Committee.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/